I am intrigued by the genealogies of our leading scientists, since their mentors too were often preeminent scientists. Earlier postings noted the example of Jonas Salk, who did postgraduate studies under Thomas Francis; one of the great pioneers of medical virology, perhaps best known for developing the first influenza vaccine (1, 2). James Watson, who did his doctoral studies in Salvatore Luria’s laboratory, and Renato Dulbecco, who trained under both Luria and Max Delbruck (3), are other examples. In fact, Watson and Dulbecco shared a lab bench in Luria’s lab. Howard Temin did his doctoral (and postdoctoral studies too) in Dulbecco’s lab (4). And Delbruck, who hugely influenced the new science of molecular biology, did his doctoral studies under Max Born, the 1954 Nobel Laureate in physics. Moreover, Delbruck later served as an assistant to Lisa Meitner (5).

Important research paths were undertaken, and major contributions were made, which resulted from less formal interactions between budding young scientists and top scientists of the day. Howard Temin’s chance encounter with Harry Rubin, while on a mission to Dulbecco’s lab, is a case in point (4).

Our last posting told how Louis Pasteur came within a whisker of adding the discovery of viruses to his list of extraordinary achievements (6). Robert Koch played a part in that story for developing his famous postulates, which provided the standard for demonstrating that a particular microbe causes a particular disease.

The Pasteur article also noted that in 1898 Friedrich Loeffler and Paul Frosch isolated the foot and mouth disease virus; the first virus isolated from animals. However, the piece did not point up that Loeffler had trained under Robert Koch. Also, it did not underscore the special significance of what Loeffler and Frosch achieved. In brief, by the 1890s Dmitry Ivanovsky and Martinus Beijerinck had independently discovered that the agent responsible for tobacco mosaic disease passes through bacterium-proof filters. Nevertheless, neither Ivanovsy nor Beijerinck appreciated the implication of their observation. Ivanovsky believed his filters might be defective, while Beijerinck thought the disease was caused by a “living liquid.” In contrast, Loeffler and Frosch, in addition to isolating the first virus that is pathogenic in animals, also carefully considered all possible explanations for their experimental findings, and then were the first to conclude the existence of a kind of microbe too small to be retained by bacterium-proof filters, and too small to be seen under a microscope, and that will not grow on laboratory culture media. They also correctly predicted that smallpox, cowpox, cattle plague, and measles are similarly caused by a “filterable virus.”

Loeffler made another major discovery, fourteen years earlier, in 1884, when he used his mentor’s postulates to identify the bacterium that causes diphtheria, Corynebacterium diphtheriae. Importantly, Loeffler also discovered that when he injected C. diphtheriae into animals, the microbe did not need to spread to the tissues it damaged. This observation led Loeffler to propose the bacteria were secreting a poison or toxin that spread to the remote sites and caused disease there.

Loeffler’s idea of a toxin was a new concept that subsequently was confirmed by Emile Roux, who had been Louis Pasteur’s assistant (6). Using bacterium-proof filters developed by Charles Chamberland in Pasteur’s lab, Roux showed that injecting animals with sterile filtrates of C. diphtheriae cultures caused death with a pathology characteristic of actual diphtheria. Roux was also a co-founder of the Pasteur Institute, where he was responsible for the production of diphtheria anti-toxin; the first effective diphtheria therapy. See Aside 1.

[Aside 1: Earlier, Roux suggested the approach Pasteur used to generate attenuated rabies virus for the Pasteur rabies vaccine (aging spinal cords from rabbits that succumbed to experimental rabies infections of their spinal cords). Roux later withdrew from the rabies project because of a disagreement with Pasteur over whether the rabies vaccine might be safe for use in humans (6).]

So, Loeffler and Roux trained under Koch and Pasteur, respectively. But why might toxin production by C. diphtheriae interest virologists. Well, in 1951, Victor Freeman at the University of Washington showed that the lethal toxins produced by C. diphtheriae (and by Clostridium botulinum as well) are the products of lysogenic bacteriophage carried by the bacteria. This was shown by the finding that avirulent strains of these bacteria became virulent when infected with phages that could be induced from virulent strains. So, are diphtheria and botulism due to bacteria or to viruses? Our chain of genealogies continues with a selective history of lysogeny.

Almost from the beginning of phage research (bacteriophage were discovered independently by Frederick Twort in Great Britain in 1915 and by Félix d’Hérelle in France in 1917), some seemingly normal bacterial cultures were observed to generate phage. Initially, this phenomenon was thought to be a sign of a smoldering, steady state kind of persistent phage infection. Then, during the 1920s and 1930s, the French bacteriologists, Eugene Wollman and his wife Elizabeth, working together on Bacillus megatherium at the Pasteur Institute, provided evidence that instead of a steady state infection, the phage actually enter into a latent form in their host cells; a form in which they might be harmlessly passed from one cell generation to the next. [Considering the state of knowledge back then, note the insightfulness of Eugene Wollman’s 1928 comment, “the two notions of heredity and infection which seemed so completely distinct and in some ways incompatible, . . . almost merge under certain conditions.”] See Aside 2.

[Aside 2: Since some bacterial strains would, on occasion, spontaneously undergo lysis and release bacteriophage, the cryptic bacteriophage they carried were called “lysogenic.” Thus, it is a bit odd that “lysogeny” eventually came to refer to the temperate relationship between these phages and their host cells.]

In the late 1930s, the Wollmans developed a close friendship with Andre Lwoff, their new colleague at the Pasteur Institute. The Wollmans introduced Lwoff to their ideas about lysogeny, but, as Lwoff confesses, he was not then impressed by bacteriophage (7).

The Nazi occupation of Paris during the Second World War began in 1940. From then on, the Jewish Wollmans were prevented from publishing their research findings. Nevertheless, they continued their research at the Pasteur Institute until 1943, when they were seized by the Nazis and sent to Auschwitz. They never were heard from again. Their friend, Lwoff, grieved their loss and became active in the French resistance, gathering intelligence for the Allies, while also hiding downed American airmen in his apartment.

After the war, Lwoff received several honors from the French government for his efforts against the Nazis. He also returned to his research at the Pasteur Institute, studying the genetics of Moraxella; a bacterial pathogen of the human respiratory tract. Because of his work as a microbial geneticist, he was invited to the 1946 Cold Spring Harbor Symposium, where he met Max Delbruck. And as happened to others, meeting Delbruck resulted in Lwoff being seduced by bacteriophage.

Andre Lwoff

Back in Paris, Lwoff’s passionate interest in phages was heightened further by discussions with Jacques Monod, a friend of Max Delbruck, and Lwoff’s neighbor in the attic of the Pasteur Institute. Although Monod was Lwoff’s junior colleague (in fact, it was Lwoff who first stirred Monod’s interest in microbiology), Lwoff’s conversations with the future Nobel Laureate resulted in Lwoff becoming intensely fascinated by lysogeny, which he began to study in 1949 (7).

Because of Lwoff’s earlier friendship with the Wollmans, he chose to study a lysogenic strain of B. megatarium. And, making use of techniques he learned from Renato Dulbecco during a brief stint at Cal Tech, he was able to follow a single lysogenic bacterium, which enabled him to observe that a bacterium could go through multiple rounds of replication without liberating virus. What’s more, he discovered that the phages are released in a burst when the cell lyses, thereby dispelling the still current notion that phages are liberated continuously by lysogenic bacteria. Furthermore, Lwoff showed that lysogenic bacteria usually do not contain phage particles, since none are detected when the cells are experimentally lysed with lysozyme; confirming the earlier (1937) findings of the Wollmans.

Lwoff went on to show that temperate phage genomes are maintained in a previously unknown integrated state in their host cell, and he gave the integrated phage genomes a name, “prophage.” He also discovered, unexpectedly, that irradiating lysogenic bacteria with ultraviolet light could induce the temperate phages to emerge from their latent state, and then replicate in, and lyse their host cells. And, he discovered that the phages lyse their host bacterial cells by producing enzymes that destroy bacterial cell walls.

Prophage

Lwoff’s elucidation of the fundamental nature of lysogeny in bacteria would later provide a paradigm for the DNA tumor viruses, the herpesviruses, the oncogenic retroviruses, and HIV. He was awarded a share of the 1965 Nobel Prize for physiology or medicine for his lysogeny research. He shared the award with his fellow Pasteur Institute scientists, François Jacob and Jacques Monod, who received their awards for their pioneering studies of gene regulation in E. Coli.

A rather intriguing aspect of this story is that Lwoff was joined in his research on lysogeny at the Pasteur Institute by Elie Wollman; the son of Eugene and Elizabeth. Elie, born in 1917, escaped from the Nazis in Paris in 1940 and worked in the French resistance as a physician. In 1946, after the war, he came to the Pasteur Institute, where he took its microbiology course and then became Lwoff’s research assistant. Then, in 1947, Elie too happened to meet Max Delbruck (in Paris in this instance) and was invited to join the Cal Tech phage group, where he spent the next two years. See Aside 3.

Elie Wollman

[Aside 3: By the early 1940s, the then young Cal Tech “phage group,” headed by Max Delbruck, was on its way to becoming the World’s great center for phage research (5). However, the American group had little interest in lysogeny, since Delbrück neither believed in it, nor saw its importance. Instead, Delbruck was totally committed to the study of lytic phages. Then, during the late 1940s, Delbruck began to lose interest in molecular biology and looked for new research directions. When he thought of turning his attention to brain function, he asked his group to put together a series of seminars based on papers written by prominent neuroscientists of the day. Elie Wollman was the only member of the Cal Tech group who declined to participate in that endeavor, since he was totally committed to bacteriophage. Moreover, Elie was the one who finally convinced Delbruck that “such a thing as lysogeny does exist (7).”

Elie himself tells us that when he looked into a bibliographical index at Cal Tech, he came across an index card referring to his parent’s 1937 paper, which reported their finding that lysogenic cells contain a non-infectious form of the phage (8). “Delbruck’s comment on the card was “Nonsense.”]

After Eli’s two-year stint with Delbruck in Pasadena, he returned to the Pasteur Institute. Meanwhile, Francois Jacob had come to the Institute in the hope of beginning a research career in genetics under the tutelage of either Lwoff or Monod. Before that, in 1940, Jacob, who also was Jewish, left medical school in occupied France to join Free French Forces in London. He then served as a medical officer in North Africa, where he was wounded, and was later severely wounded at Normandy in August 1944, ending his dream of becoming a surgeon.

Francois Jacob

Initially, Jacob was spurned by both Lwoff and Monod, but was finally taken on by Lwoff, who suggested that he, Jacob, start work on “the induction of the prophage.” Jacob confesses he had no idea what that meant, but he accepted the project. Thus it came to pass that Francois Jacob and Elie Wollman established a particularly close and friendly collaboration, in which they turned their attention to the lambda prophage of E. coli. Their initial goal was to clarify the events of bacterial conjugation so that they might then understand the phenomenon whereby a temperate phage carried by a lysogenic bacterium is activated to undergo vegetative replication when that bacterium conjugates with, and transfers its integrated phage genome to a non-lysogenic bacterium.

To accomplish their goal, Wollman and Jacob began with experiments to locate the lambda genome on the chromosome of the lysogenic cell, and to follow its transfer during conjugation into a non-lysogenic recipient cell. A key feature of their experimental approach was conceived by Wollman (8). It was simply to interrupt conjugation between a lysogenic donor (Hfr) cell and a non-lysogenic recipient (F-minus) cell, at various times, by using a kitchen blender to break the mating cells apart. Using the blender to interrupt conjugation, and also using bacterial strains in which the recipient bacteria contained a set of mutations, and plating the mating mixture on selective media, Wollman and Jacob were able to measure the length of time required for each of the corresponding wild-type genes to be transferred from the Hfr donor cells to the F-minus recipient cells. Indeed, the time intervals between the appearances of each wild type gene in the recipient cells directly correlated with the distances between the genes, as independently determined by recombination frequencies. Thus, the interrupted mating approach gave Wollman and Jacob a new means to construct a genetic map of the bacterium, while also enabling them to locate the integrated phage genome on that map. Their experimental approach also allowed Wollman and Jacob to establish that, during conjugation, the donor cell’s genome is transferred linearly to the recipient cell. [The designation “Hfr” was coined by William Hayes because Hfr strains yielded a high frequency of recombinants when crossed with female strains.]

Importantly, Wollman and Jacob’s study of the activation of a lambda prophage when it enters a non-lysogenic F-minus recipient (a phenomenon they called “zygotic induction”), showed that the temperate state of the lambda prophage is maintained by some regulatory factor present in the cytoplasm of a lysogenic bacterium, but which is absent from a non-lysogenic one. It led to the discovery of a “genetic switch” that regulates the activation of the lysogenic bacteriophage, and of a phage-encoded repressor that controls the switch. These findings are among the first examples of gene regulation, and are credited with generating concepts such as the repressor/operator, which were firmed up by Jacob and Monod in their Nobel Prize-winning studies of the E. coli lac operon. See Aside 4.

[Aside 4: At the time of Wollman and Jacob’s interrupted mating experiments, kitchen blenders had not yet made their way to European stores. Eli was aware of these appliances only because of his earlier stint at Cal Tech. He bought a blender for his wife before returning to France, and then “borrowed” it for these experiments.]

Wollman and Jacob went on to demonstrate that the fertility or F factor, which confers maleness on the donor bacteria, can exist either in an integrated or an autonomous state. Indeed, this was the first description of such a genetic element, for which they coined the term “episome;” a term now largely replaced by “plasmid.”

Wollman and Jacob also determined that the E. coli chromosome is actually a closed circle. The background was as follows. Only one F factor is integrated into the chromosome of each Hfr strain, and that integration occurs at random. And, since the integrated F factor is the origin of the gene transfer process from the Hfr cell to the F-minus cell, interrupted mating experiments with different Hfr strains gave rise to maps with different times of entry for each gene. However, when these time-of-entry maps were taken together, their overlapping regions gave rise to a consistent circular map. The discovery of the circular E. coli chromosome was most intriguing, because all previously known genetic maps were linear. See Aside 5.

[Aside 5: The bacterial strain used by Wollman and Jacob in their study of zygotic induction was, in fact, the original laboratory strain of E. coli (i.e. E. coli K12) that was isolated in1922 from a patient with an intestinal disorder. In 1951, Esther Lederberg discovered that K12 is lysogenic. The discovery happened when she accidentally isolated non-lysogenic or “cured” derivatives of E. coli K12 that could be infected by samples of culture fluid from the parental K12 strain, which sporadically produced low levels of phage. Esther gave the lysogenic phage its name, lambda.

Esther was the wife of Joshua Lederberg, who received a Nobel Prize in 1958 for discovering sexual conjugation in bacteria, and the genetic recombination that might then ensue. Prior to Lederberg’s discoveries, genetic exchange and recombination were not believed to occur in bacteria. Lederberg’s Nobel award was shared with George Beadle and Edward Tatum (the latter was Lederberg’s postdoctoral mentor) for their work in genetics.

Joshua Lederberg, working with Norton Zinder (9), also discovered transduction, whereby a bacterial gene can be transferred from one bacterium to another by means of a bacteriophage vector. And, working together with Esther, Joshua discovered specialized transduction, whereby lambda phage transduces only those bacterial gene sequences in the vicinity of its integration site on its host chromosome. Esther and Joshua also worked together to develop the technique of replica plating, which enabled the selection of bacterial mutants from among hundreds of bacterial colonies on a plate and, more importantly perhaps, to provide direct proof of the spontaneous origin of mutants that have a selective advantage.]

In 1954 Elie Wollman was appointed a laboratory head in his own right at the Pasteur Institute. He retired from research in 1966 to become vice-director of the Institute, which he then rescued from a severe financial crisis. He continued to serve in that role for the next 20 years, while garnering numerous prestigious awards for his research and service.

Francois Jacob earned his doctorate in 1954 for his lysogeny studies. Then, realizing that he and Jacques Monod, his senior neighbor in the Pasteur Institute attic, were actually studying the same phenomenon, gene repression, he entered into a hugely productive collaboration with Monod that led to the elucidation of the genetic switch that regulates beta-galactosidase synthesis in E. coli (9). Their collaboration established the concepts of regulator genes, operons, and messenger RNA, for which they shared in the 1965 Nobel Prize for physiology or medicine, as noted above. See Asides 6 and 7.

Jacques Monod

[Aside 6: One of Jacob and Monod’s first experiments was the famous 1957 PaJaMa experiment, carried out in collaboration with Arthur Pardee, who was then on sabbatical at the Pasteur Institute. In brief (for aficionados), a Lac-positive, Hfr strain was grown in an inducer-free media, and then mated, still in an inducer-free media, with a Lac-minus, F-minus strain. (Note that the deletion in the Lac-minus, F-minus strain included the LacI gene, which encodes the yet to be discovered lac repressor.) As expected, in the absence of inducer, no beta-galactosidase is detected initially. But, after the donor DNA sequence, which bears the normal Lac genes (including LacI), is transferred to the Lac-minus recipient, it initially finds no repressor in the recipient cell and begins to synthesize beta-galactosidase. Then, as the donor cell’s lac repressor gene begins to be expressed in the recipient cell, in the inducer-free media, expression of the donor cell’s beta-galactosidase gene ceases. The PaJaMa experiment thus showed that the genetic regulation of enzymatic induction depends on a previously unknown regulatory molecule, the repressor.

Notice the similarity between the rationale for the PaJaMa experiment and that of the earlier Wollman and Jacob experiment on zygotic induction. In each instance, a process regulated by a repressor is suddenly in the repressor-free environment of a recipient cell.]

[Aside 7: In June of 1960, Francois Jacob, Matt Meslson, and Sidney Brenner came together in Max Delbruck’s Cal Tech lab to carry out an experiment that confirmed the existence of messenger RNA. The key to the experiment was their ability to distinguish ribosomes present in the cell before infection from ribosomes that might have been made after infection. They cleverly did that by incorporating heavy isotopes into ribosomes before infection, so that they might be separated in a density gradient from ribosome made after infection. Then, they showed that RNA produced by T2 phage in E. Coli associates with ribosomes that were synthesized by the cell entirely before infection. Furthermore, the new phage-specific RNA directs the synthesis of phage-specific proteins on those “old” ribosomes. I vote for this experiment as the most elegant in the entire history of molecular biology (11).]

Incidentally, during the Nazi occupation of Paris, Monod too was active in the French Resistance, eventually becoming chief of staff of the French Forces of the Interior. In that capacity, he helped to prepare for the Allied landings in Normandy. Monod and Jacob each received France’s highest honors for their wartime service.See Aside 7.

[Aside 7: I am singularly intrigued by the experiences of Andre Lwoff, Elie Wollman, Francois Jacob, and Jacques Monod during the Second World War. References 3 and 5 recount the wartime experiences of Renato Dulbecco and of Max Delbruck, and of other great scientists of the time. Other posts on the blog give accounts of virologists courageously placing themselves in harm’s way under different circumstances. Examples include pieces featuring Ciro de Quadros, Carlo Urbani, Peter Piot, and Walter Reed.]

References:

(1) Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posted on the blog March 27, 2014.

(2) Ernest Goodpasture and the Egg in the Flu Vaccine, Posted on the blog November 25, 2014.

(3) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, Posted on the blog, December 4, 2013.

(4) Howard Temin: “In from the Cold,” Posted on the blog December 16, 2013
(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.

(6) Louis Pasteur: One Step Away from Discovering Viruses, Posted on the blog January 7, 2015.

Louis Pasteur (1822-1895) is the subject of our first posting of the New Year. Pasteur was history’s greatest microbiologist and, perhaps, its most famous medical scientist. Pasteur was also an early figure in the history of virology for his 1885 discovery of a rabies vaccine; only the second antiviral vaccine and the first attenuated one (see Aside 1). However, the main point of this tale is that Pasteur let pass an especially propitious opportunity to discover that the rabies agent is one of a previously unrecognized class of microbes; a class that is fundamentally different from the already known bacteria. Its members are submicroscopic and grow only inside of a living cell. Pasteur was just one step away from discovering viruses.

Louis Pasteur

[Aside 1: Attenuation is the conversion of a pathogenic microbe into something that is less able to cause disease, yet is still able to induce immunity. Edward Jenner’s 1798 smallpox vaccine, the world’s first vaccine, as well as the first antiviral vaccine, was not based on the principle of attenuation. Instead, it contained live, unmodified cowpox virus. Although hardly understood in Jenner’s day, his smallpox vaccine worked because cowpox, which is not virulent in humans, is immunologically cross-reactive with smallpox. Thus, the relatively benign cowpox virus induced immunity against the related, deadly smallpox virus (1).]

The distinctive nature of viruses would first begin to be revealed in 1887 by a scientist of much less renown than Pasteur; the Russian microbiologist Dmitry Ivanovsky. The virus concept would be further advanced in 1898 by the accomplished Dutch botanist Martinus Beijerinck (2). In any case, to better appreciate how anomalous it was that Pasteur did not discover viruses, we review the greatness of his earlier achievements. After that, we consider the opportune circumstance that he let go by.

Pasteur was a chemist by background. Thus, his first major scientific discovery, at 26 years of age, was as a chemist. It was his 1847 discover of molecular asymmetry; that certain organic molecules exist in two alternative molecular structures, each of which is the mirror image of the other. Additionally, pairs of these asymmetric molecules are chemically indistinguishable from each other, and balanced mixtures of them rotate the plane of polarized light.

Pasteur’s discovery of molecular asymmetry was one of the great discoveries in chemistry. Yet his research would take on a momentous new focus when he began to investigate the chemistry of fermentations. This new course was inspired by the fact that while asymmetric molecules are not generated in the laboratory, they are found in the living world. And, since asymmetric molecules are found among fermentation products, Pasteur hypothesized that fermentation is a biological process, which he proceeded to demonstrate in 1857, basically by showing that fermentation products did not arise in nutrient broth if any microbes that might have been present were either killed by heating or removed by filtration. What’s more, he showed that specific fermentations are caused by specific microorganisms. Additionally, he discovered that fermentation is usually an anaerobic process that actually is impaired by oxygen; a phenomenon known as the “Pasteur effect.” And, he put forward the notion of aerobic versus anaerobic microbes.

Pasteur put his experience studying fermentations to practical use when he came to the rescue of the French wine industry, which was on the verge of collapse because of the wine becoming putrefied. Pasteur showed that the problem was due to bacterial contamination, and then showed that the putrefaction could be prevented by heating the wine to 50 to 60 °C for several minutes; a procedure we now refer to as pasteurization. Wines are seldom pasteurized today because it would kill the organisms responsible for the wines maturing. But, as we know, pasteurization is applied to many contemporary food products, especially milk. Pasteur also aided the beer industry by developing methods for the control of beer fermentation.

Pasteur’s study of fermentations led to an experiment of historic significance for biology in general. In the 1860s, the ancient notion that life can arise spontaneously from nonliving materials, such as mud or water, was still widely believed. The emerging awareness of microbes in the 1860s did not change this belief. Instead, it led to the idea that fermentations and putrefactions result from the spontaneous generation of microbes. In 1862, Pasteur unequivocally dispelled this belief by a simple yet elegant experiment in which he made use of a flask that had a long bending neck that prevented contaminants from reaching the body of the flask. If the broth in the flask was sterilized by boiling, and if the neck remained intact, then the broth remained sterile. But, if the neck of the flask was broken off after the boiling, then the broth became opaque from bacterial contamination.

Taken alone, Pasteur’s achievements that are enumerated above would have been sufficient to have ensured his lasting fame. Nevertheless, Pasteur’s greatest successes were yet to come. In 1867 he put forward the “germ theory of disease.” By this time, the existence of a variety of microorganisms, including bacteria, fungi, and protozoa, was already well established. Pasteur’s new proposal, that microorganisms might produce different kinds of diseases, was inspired by his earlier experimental findings that different microorganisms are associated with different kinds of fermentations, and by his 1865 finding that a microorganism was responsible for a disease in silkworms that was devastating the French silk industry.

After Pasteur proposed his germ theory of disease, Robert Koch (another giant in the history of medical microbiology) established that anthrax in cattle is caused by a specific bacterium, Bacillus anthracis. Koch had taken a sample from diseased cattle and then used his new method for isolating pure bacterial colonies on solid culture media to generate a pure culture of B. anthracis. Next, he inoculated healthy animals with a portion of the pure culture. The healthy animals then developed anthrax. Finally, he re-isolated B. anthracis from the inoculated animals. This sequence of isolation, infection, and re-isolation constitutes Koch’s famous postulates, which provide the experimental basis for establishing that a specific microorganism is responsible for a specific disease.

Even after Pasteur confirmed Koch’s anthrax findings in 1877, some members of the medical establishment still rejected the germ theory of disease, mainly because Pasteur was a chemist by background, rather than a physician. Nevertheless, Joseph Lister, an English surgeon, admired Pasteur’s work on fermentation and was impressed by Pasteur’s disproving of spontaneous generation. Based on Pasteur’s demonstration of the ubiquity of airborne microorganisms (another of his noteworthy achievements), Lister reasoned that infections of open wounds are due to microorganisms in the environment. The aseptic techniques that Lister then introduced were responsible for dramatically reducing infections during surgery.

The following is one of my favorite parts of this story. In 1879, Pasteur made his first important contribution to vaccinology, when he discovered, by accident, that he could attenuate the bacterium responsible for chicken cholera (now known to be a member of genus Pasteurella), and then use the attenuated microbe as a vaccine. It happened as follows. Pasteur instructed his assistant, Charles Chamberland, to experimentally inject chickens with the cholera bacterium so that he, Pasteur, might observe the course of the disease. Then, just before a summer holiday break, Pasteur directed Chamberland to inject the chickens with a fresh culture of the bacteria. Chamberland may have been preoccupied with thoughts of the upcoming holiday, because he forgot to inject the chickens before leaving. When he returned a month later, he carried out Pasteur’s instructions, except that he injected the chickens with the now aged bacteria. What happened next was most important. The chickens that were inoculated with the aged culture developed only a very mild form of the disease. After that, Pasteur had Chamberland inject those same chickens with freshly grown, presumably virulent bacteria. The chickens still did not develop disease.

It is not clear why Pasteur instructed Chamberland to inoculate the freshly grown culture into the chickens that earlier had received the aged culture. Perhaps it was an accident, or perhaps Pasteur saw an opportunity to carry out a possibly interesting experiment. (The chickens had survived a mild infection by the aged culture. Might they now be resistant to freshly grown virulent bacteria?) In any case, Pasteur repeated the entire sequence of inoculating the chickens with an aged culture and then challenging them with a fresh culture. The outcome was the same as before.

Pasteur correctly surmised that the aging process (actually, oxidation by exposure to air) had attenuated the bacteria. And, he learned by experimentation that the virulence of the cholera microbe could be reduced to any desired extent by controlling its exposure to air. Most importantly perhaps, he discovered that the attenuated bacteria could induce resistance to the virulent bacteria and, consequently, could be used as a vaccine. Pasteur’s chicken cholera vaccine was the first vaccine deliberately created in a laboratory. What’s more, it was the first attenuated vaccine. See Aside 2.

[Aside 2: During the years that Pasteur was carrying out his vaccine studies, nothing was known regarding the physiological basis of immunity, or the determinants of virulence, or of mutations, or the underlying mechanism of attenuation that changed a deadly microbe into a harmless one that still could induce immunity. Considering the intellectual milieu in which Pasteur carried out his investigations, it is all the more remarkable that he achieved so much. And while Pasteur’s interpretations for how his attenuated vaccines worked were far from accurate, they are still impressive for their plausibility. Initially, he thought that the attenuated organisms might simply compete with the virulent organisms for a limited availability of nutrients in the host. Later, he thought that the attenuated organisms might release a toxin that blocked growth of the virulent organisms. The notion, that the host might actually initiate its own defense, began to emerge in 1890 when Emil von Behring and Shibasaburo Kitasato discovered that a host factor neutralized the diphtheria toxin. Kitasato then put forward the theory of humoral immunity, proposing that a host serum factor could neutralize a foreign antigen. In 1891 Paul Ehrlich used the term “antibody” for the first time, in reference to those serum factors.]

This account of the cholera vaccine brings to mind Pasteur’s famous remark, “Chance only favors the prepared mind.” Yet in the context of our larger story, it is an ironic statement, considering that Pasteur later missed an auspicious opportunity to discover viruses. But, before getting to that, we briefly note Pasteur’s work on his anthrax vaccine.

In 1879 Pasteur began to develop an anthrax vaccine, which, like the cholera vaccine, would be based on his principle of attenuation. And, as in the case of the cholera vaccine, Pasteur attenuated the anthrax bacillus by exposing it to oxygen. [History records that Pasteur and his assistants developed a second approach to attenuate the anthrax bacillus, based on their discovery that when the bacilli are cultivated at 42 or 43 degrees centigrade, they do not develop the endospores that are necessary to cause a virulent infection.] In 1881 Pasteur carried out a dramatic public demonstration of the effectiveness of his air-oxidized anthrax vaccine in livestock, causing many doubters to accept the validity of his work. See Aside 3 and the end note.

[Aside 3: Currently, the only FDA-licensed anthrax vaccine for use in humans is BioThrax, produced by Emergent BioDefense Operations Lansing Inc. BioThrax is generated from an avirulent, nonencapsulated mutant of B. anthracis. It does not contain any living organisms. As suggested by the name of the manufacturer, BioThrax was produced mainly for the U.S. Department of Defense, for use in case B. Anthracis might be used as a biological weapon. Thus, BioThrax is not available to the public. People who are exposed to B. anthracis are now treated with antibiotics (e.g., ciprofloxacin and doxycycline).]

Pasteur turned his attention to rabies in1880, when the problem of rabid dogs in Paris was getting out of hand. Once again Pasteur sought to develop a vaccine, and once again he wanted to apply the principle of attenuation. But, early on, he found that he could not grow the rabies agent in pure culture. Thus, he was not able to isolate the rabies agent. Moreover, he would need to devise new procedures if he was to grow and attenuate it. His solution was to develop methods for cultivating the rabies agent in the spinal cords of live rabbits. His method for attenuation was then suggested by his assistant, Emile Roux, who had been studying survival of the rabies agent in pieces of rabbit spinal cord that he suspended inside a flask. Following Roux’s example, Pasteur attenuated the rabies agent by air-desiccating spinal cords taken from experimentally infected rabbits that earlier had died of rabies. Each successive day of desiccation resulted in greater attenuation of virulence, such that an extract from a spinal cord aged for 14 days could no longer transmit the disease. What’s more, those extracts could be used as inoculums that prevented rabies in dogs that later were challenged with the virulent microbe.

Pasteur, himself, took saliva samples from rabid dogs. In one such incident, he used a glass tube to suck up a few drops of deadly saliva from the mouth of a mad, squirming bulldog that was held down on a table by two assistants. The assistants wore heavy leather gloves.

Here is another of my favorite parts of this story. In 1885, nine-year-old Joseph Meister was bitten repeatedly by a rabid dog. Young Joseph’s desperate mother then brought her son to Pasteur, hoping that he might help Joseph. But, any attempt by Pasteur to treat the boy was sure to provoke controversy. Pasteur was not a medical doctor. Moreover, his rabies vaccine had never been successfully used in humans. Furthermore, attenuation and vaccination were still new and contentious concepts. For these reasons, Pasteur rejected many earlier requests for help from people in France, and from abroad as well. But, in Joseph’s case, Pasteur relented, convinced that the boy would die if he did not intercede.

Pasteur gave young Joseph a series of 13 injections, one each day, in which each successive injection contained a less-attenuated (stronger) virus. Pasteur dreaded inoculating Joseph with the last shot in the series; a one-day-old vaccine that was strong enough to kill a rabbit. Emile Roux wanted no part in this episode and, in fact, withdrew from the rabies study because of it. But, Joseph never developed rabies, and millions of people subsequently received Pasteur’s anti-rabies treatments. [Pasteur’s attenuated rabies vaccine may not have been entirely safe for humans. Modern rabies vaccines are generally killed virus vaccines, prepared by chemically inactivating tissue culture lysates.] See Asides 4 and 5.

[Aside 4: Post-infection rabies vaccination works and, indeed, is necessary because (for reasons that are still not entirely clear) the human immune response against a natural rabies infection is not able to prevent the virus from reaching the central nervous system, at which point the infection is invariably fatal. Importantly, the incubation period between the time of the bite and the appearance of disease can be more than several months, and is never less than two weeks. Consequently, there is a substantial window of opportunity for the vaccine to cause the virus to be inactivated at the site of the bite.]

[Aside 5: In 1888, Emile Roux, working at the Pasteur Institute (see below), would confirm the existence of the diphtheria toxin by showing that injecting animals with sterile filtrates of liquid cultures of Corynebacterium diphtheriae caused death with a pathology characteristic of actual diphtheria.]

Pasteur worked hard to isolate the rabies agent, but he wrongly presumed that he should be able to grow it in pure culture. Finally, in 1884, he conceded that he had not been able to isolate and cultivate the rabies agent in a laboratory media. So, might that failure alone have been sufficient to cause Pasteur to think of the rabies agent in new terms? Perhaps not, since, at the time, the inability to cultivate a microbial pathogen was assumed to be a laboratory failure, rather than a reason to hypothesize that that the agent was something other than a bacterium. [Even with the eventual awareness of the uniqueness of viruses, the inability of virologists to cultivate viruses outside of an animal would remain a mystery, as well as an obstacle, well into the early 1930s (3).]

Pasteur also got sidetracked while trying to isolate the rabies agent. In 1880 he injected a rabbit with the saliva of a child who died of rabies. He then examined the blood of the rabbit after it too succumbed to rabies. Using his microscope, Pasteur in fact saw a microbe in the rabbit’s blood, which he thought might be the rabies agent. However, he later found the same microbe in the saliva of normal children. Ironically, this microbe, which Pasteur at first thought might be the rabies agent, was actually Pneumococcus pneumoniae, a major bacterial pathogen that was correctly identified several years later by Albert Frankel. Thus, Pasteur missed the opportunity to identify a bacterial pathogen that is much more important in humans than rabies virus. Moreover, and importantly, Pasteur never did see the actual rabies agent under his microscope. Thus, he was aware that the rabies agent might be unusually small in comparison to the usual bacteria.

Here is another bit of irony. The item (apparatus?) that initially played the key role in distinguishing viruses from bacteria was invented in Pasteur’s laboratory. It was the unglazed terra cotta filter, conceived by Charles Chamberland, which he used to provide a good supply of sterile water for Pasteur’s lab. Chamberland allegedly developed these bacterium-proof filters while experimenting with a broken clay pipe that he bought from his tobacconist.

Bearing in mind that Pasteur was never able to grow the rabies agent in pure culture, and that he never saw the rabies agent under his microscope, might he have thought that it might be a submicroscopic infectious agent that is different from bacteria in some fundamental way? I have not come across any definitive answer to that question. But, I feel safe to say that it is unlikely that anyone other than Pasteur might have seriously considered that possibility. Regardless, Pasteur did not take the next logical step, which would have been to see if the rabies agent might pass through Chamberland’s filters. Had he done so, he could have isolated the rabies agent from the rabbit spinal cords, and he would have discovered “filterable viruses” (see below).

That crucial step was taken for the first time in 1887 by the Russian bacteriologist, Dmitry Ivanovsky, who used Chamberland filters in his investigations into the cause of tobacco mosaic disease. Ivanovsky could not propagate the tobacco mosaic agent (later known as the tobacco mosaic virus) in pure culture. However, because of his finding that the agent could actually pass through Chamberland’s filters, Ivanovsky is sometimes credited for discovering viruses. Yet Ivanovsky did not accept his own results. He still presumed that the disease was caused a bacterium, and he thought that the filters were defective or, instead, that the disease was due to a toxin produced by the bacterium.

In 1898, Martinus Beijerinck, unaware of Ivanovsky’s earlier work, also could not see or cultivate the tobacco mosaic agent. In addition, he too found that the agent passed through Chamberland filters. Beijerink expected, and perhaps even hoped that the filters would remove the agent from diseased plant extracts, so that he might prove it to be a bacterium. But despite his possible disappointment, Beijerinck went one major step further. He demonstrated that the filtered sap from a diseased plant did not lose its ability to cause disease after being diluted by repeated passage through new healthy plants. Consequently, the filterable agent was replicating in the plant tissue and, thus, could not be a toxin.

Little is recorded about Ivanovsky, aside from his four-page report on the tobacco mosaic disease (see Aside 6). In contrast, Beijerinck was a major scientist, who made numerous important contributions, including the discovery of nitrogen-fixing bacteria and bacterial sulfate reduction (4). Yet even Beijerinck found it difficult to conceive that the filterable, incredibly small, submicroscopic tobacco mosaic agent might be particulate in nature. Instead, he famously described it as a “contagious living fluid.” Nonetheless, Beijerinck, a botanist by background, is often considered to be the first virologist.

[Aside 6: Ivanovsky’s four-page paper would be unremarkable if it were not for the single sentence, “Yet I have found that the sap of leaves attacked by the mosaic disease retains its infectious qualities even after filtration through Chamberland filters.”]

Pasteur was probably unaware of Ivanovsky’s findings, and he did not live long enough to know of Beijerinck’s. So, we do not know what he might have made of their results. Regardless, Pasteur remained one step away from making these discoveries himself.

In 1898, after the announcement of Beijerinck’s findings, Friederich Loeffler and Paul Frosch isolated the foot and mouth disease virus; the first virus isolated from animals. Next, in 1901, in Cuba, U.S. Army doctor Walter Reed isolated yellow fever virus (5); the first pathogenic virus of humans to be isolated. In 1903, Paul Remlinger, working at the Constantinople Imperial Bacteriology Institute, filtered and then isolated rabies virus. Despite these early achievements, it was not until 1938 that the development of the electron microscope made it possible to resolve that viruses are indeed particulate, rather than liquid in nature. See Aside 7.

[Aside 7: The term “virus” indeed appears in the scientific literature of Pasteur’s day. However, at that time “virus” referred to any microbe that might cause disease when inoculated into a susceptible human or animal. By the 1890s, the term “filterable virus” came into use, meaning an infectious agent which, like the tobacco mosaic virus, passed through filters that retained bacteria. But, bearing in mind that there was not even a consensus regarding the identity of the genetic material until the early 1950s, there would be no clear understanding of viruses until then. In fact, the classic, early 1950s blender experiment of Alfred Hershey and Martha Chase, which featured bacteriophage T4, played a key role in establishing DNA as the genetic material, while also elucidating the essentials of virus replication (2).]

In 1887 Louis Pasteur founded the Institute in Paris that bears his name. A minor irony is that the Pasteur Institute was founded as a rabies vaccine center. The Institute has since been the site of numerous major discoveries in infectious diseases. But we underscore here that it was the site where, in 1910, Constantin Levaditi and Karl Landsteiner demonstrated that poliomyelitis is caused by a filterable virus, and where Félix d’Herelle in 1917 discovered bacteriophages. And it was also the site where, in 1983, Luc Montagnier and Françoise Barré-Sinoussi were the first to isolate HIV (6).

In a fitting end to our story, when Joseph Meister grew up, he became the gatekeeper of the Pasteur Institute. Meister was still minding the gate at age sixty four when, in 1940, the Nazis invaded Paris. Legend has it that when Nazi soldiers arrived at the Institute and ordered Meister to open Pasteur’s crypt, rather than surrendering Pasteur’s resting place to the Nazis, Meister shot himself (7).

Pasteur Institute: Museum and Crypt

End note:

Science historian, Gerald L. Geison, wrote a controversial revisionist account of Pasteur’s achievements, that was based on Geison’s reading of Pasteur’s laboratory notes (8). Geison undermines Pasteur’s integrity and discredits some of his major accomplishments. For example, Geison asserts that Pasteur surreptitiously used the oxidation procedure of French veterinary surgeon, Henry Toussaint, when preparing his own widely acclaimed anthrax vaccine for its public demonstration.

Max Perutz, who shared the 1962 Nobel Prize for Chemistry with John Kendrew for their studies of the structures of hemoglobin and myoglobin, reviewed Geison’s book for The New York Review of Books (December 21, 1995). Perutz’s review, entitled The Pioneer Defended, contains a vigorous rebuttal of Geison’s claims. Geison responded to Perutz’s review in the April 4, 1996 issue of The New York Review of Books. Perutz’s counter-response immediately follows.

I make note of all this because Geison’s uncertain assertions are reported as unqualified fact in some accounts of Pasteur’s work. And, while Perutz’s representations are not entirely accurate, the review, the response, and the counter-response make a very interesting read.

References:

(1) Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.

Blogs I Follow

Welcome!

I am now a retired professor emeritus of Microbiology at the University of Massachusetts. Teaching virology has been a most rewarding aspect of my career. I especially enjoyed enlivening my lectures with a variety of relevant anecdotes.

Virology Textbook

Based on my experiences teaching virology for more than 35 years, I wrote Virology: Molecular Biology and Pathogenesis (ASM Press; 2010). For info on adopting or buying this textbook, please visit the publisher site: http://www.asmscience.org/content/book/10.1128/9781555814533